|
Plant Physiol. (1998) 118: 1233-1241
A Gene Encoding Proline Dehydrogenase Is Not Only Induced by
Proline and Hypoosmolarity, but Is Also Developmentally Regulated in
the Reproductive
Organs of Arabidopsis1
Kazuo Nakashima,
Rie Satoh,
Tomohiro Kiyosue2,
Kazuko Yamaguchi-Shinozaki*, and
Kazuo Shinozaki
Biological Resources Division, Japan International Research Center
for Agricultural Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8686,
Japan (K.N., R.S., K.Y.-S.); and Laboratory of Plant Molecular Biology,
The Institute of Physical and Chemical Research (RIKEN), 3-1-1
Koyadai, Tsukuba, Ibaraki 305-0074, Japan (T.K., K.S.)
 |
ABSTRACT |
The
cDNA clone ERD5 (early
responsive to dehydration), isolated from
1-h-dehydrated Arabidopsis, encodes a precursor of proline (Pro)
dehydrogenase (ProDH), which is a mitochondrial enzyme involved in the
first step of the conversion of Pro to glutamic acid. The transcript of
the erd5 (ProDH) gene was undetectable
when plants were dehydrated, but large amounts of transcript
accumulated when plants were subsequently rehydrated. Accumulation of
the transcript was also observed in plants that had been incubated
under hypoosmotic conditions in media that contained L- or
D-Pro. We isolated a 1.4-kb DNA fragment of the putative
promoter region of the ProDH gene. The
 -glucuronidase (GUS) reporter gene driven by the
1.4-kb ProDH promoter was induced not only by
rehydration but also by hypoosmolarity and L- and
D-Pro at significant levels in transgenic Arabidopsis
plants. The promoter of the ProDH gene directs strong GUS activity in reproductive organs such as pollen and pistils and in
the seeds of the transgenic plants. GUS activity was detected in
vegetative tissues such as veins of leaves and root tips when the
transgenic plants were exposed to hypoosmolarity and Pro solutions. GUS
activity increased during germination of the transgenic plants under
hypoosmolarity. The relationship between Pro metabolism and the
physiological aspects of stress response and development are discussed.
| |
INTRODUCTION |
Osmotic or water stress caused by drought or high salinity is the
most serious factor that limits plant growth and productivity (Boyer,
1982 ). These conditions induce dehydration of plant cells, which may
trigger physiological, biochemical, and molecular responses (Shinozaki
and Yamaguchi-Shinozaki, 1996, 1997). To counteract osmotic stress,
some plants accumulate several kinds of compatible osmolytes, such as
Pro, Gly betaine, and sugar alcohols, that function as osmotica and
protect macromolecules such as proteins and membranes (Delauney and
Verma, 1993). Among the compatible solutes, Pro is the most widely
distributed osmolyte in water-stressed plants. It has been suggested to
function as a nitrogen-storage compound (Ahmad and Hellebust, 1988), as
an energy or reducing power sink (Walton et al., 1991), and as a
radical scavenger (Smirnoff and Cumbes, 1989).
The accumulation of Pro in dehydrated plants is
caused not only by the activation of Pro biosynthesis but also by the
inactivation of Pro degradation; conversely, a decrease in the level of
accumulated Pro in rehydrated plants is caused not only by the
inhibition of Pro biosynthesis but also by the activation of Pro
degradation. Genes that encode enzymes in Pro biosynthesis have been
isolated from various plants, and their expression and the functions of their products have been characterized (Delauney and Verma, 1993; Yoshiba et al., 1995, 1997; Igarashi et al., 1997). However, the molecular mechanism of Pro degradation is poorly understood.
We isolated the ProDH gene (Pro
dehydrogenase) from Arabidopsis
(Kiyosue et al., 1996) that is identical to that of
Verbruggen et al. (1996) and Peng et al. (1996). Sequence analysis of
an Arabidopsis cDNA clone, ERD5 (early
responsive to dehydration), isolated from
plants dehydrated for 1 h revealed that it encodes a protein
having homology with products of the yeast PUT1 gene and the
Drosophila melanogaster sluggish-A gene (Kiyosue et al., 1996). Their gene products are precursors of ProDH proteins (Pro oxidases: EC 1.5.99.8), which are the first enzymes involved in the
conversion of Pro to Glu. We show that the products of ERD5 cDNA are localized in the mitochondrial fraction. Fusion genes for
ERD5 and PUT1 complemented a put1
mutant of yeast, allowing put1 to grow with Pro as the
source of nitrogen. RNA gel-blot analysis demonstrated that transcripts
of the ProDH gene were undetectable when plants had been
dehydrated for 10 h, but that large amounts of the transcript
accumulated when plants were subsequently rehydrated. Elevated levels
of the transcript were also found in plants incubated in a medium that
contained Pro. These results suggest that the ProDH gene is
regulated at the transcriptional level by both dehydration and
rehydration of plants.
To investigate the regulatory mechanism for the expression of the
ProDH gene, we examined its expression by northern-blot analysis and demonstrated that the ProDH gene is
up-regulated by hypoosmolarity and Pro treatment. Then, we isolated the
promoter region of the ProDH gene and analyzed the
regulatory mechanisms in response to environmental and developmental
signals in transgenic Arabidopsis, which contained a fused gene
consisting of the ProDH promoter and the coding region of
the reporter gene for GUS. The roles of Pro dehydrogenase in stress
response, seed and pollen development, and germination are discussed.
| |
MATERIALS AND METHODS |
Plant Materials
Plants (Arabidopsis ecotype Columbia) were grown on
vermiculite beds or aseptically on GM (germination medium containing
0.09 M Suc) (Valvekens et al., 1988) containing 0.8%
Bacto-agar (Difco, Detroit, MI) for 2 to 4 weeks under continuous light
(3000 lux), as previously described (Kiyosue, 1993b, 1994; Nakashima et
al., 1997).
Stress Treatments
Arabidopsis rosette plants were subjected to dehydration on
chromatography paper (3MM, Whatman) at 60% to 70% RH and 22°C, under dim light (100 lux) (Nakashima et al., 1997). The Arabidopsis plants grown on vermiculite beds or GM agar medium were transferred to
distilled water, GM liquid medium, 0.09 M or 0.26 M L- or D-Pro, respectively, and
incubated for 1 to 24 h under dim light. In each case, the plants
were subjected to the stress treatment for varying durations and were
then immediately frozen in liquid nitrogen.
Osmolarity Measurement
The osmolarity of solutions used in northern-blot analysis was
measured at 20°C by the ONE-TEN osmometer (Fiske, Norwood, MA).
Mapping of the Transcription Start Site by Primer Extension
The primer-extension experiment was performed according to the
method of Yamaguchi-Shinozaki et al. (1989) using the
[ -32P]ATP-labeled oligonucleotide, which
corresponds to the complementary sequence upstream of the coding region
of the ProDH gene: 5 -ATAATTTCTCTTCTC-3 (complementary
to positions +66 to +80). The mRNA for the primer extension was
extracted from Arabidopsis rosette plants incubated in 0.09 M L-Pro for 2 h by the guanidine
thiocyanate/CsCl method, and was purified on an oligo(dT) column.
Isolation of RNA and RNA Gel-Blot Analysis
RNA was isolated from whole rosette plants as previously described
(Kiyosue, 1993a). Fragments of the ERD5 cDNA were labeled by
the random-primer method with [ -32P]dCTP
(Amersham) using the random-primed DNA-labeling kit from Boehringer
Mannheim. The labeled fragments were hybridized with RNA according to
standard protocols (Kiyosue et al., 1994).
Transgenic Plants
A 1.4-kb fragment of the ProDH gene upstream sequence
was prepared using double digestion with BamHI and
HindIII of the pBluescript II vector (Stratagene). It was
ligated into the BamHI and HindIII site of the
promoterless GUS expression vector pBI101.1 (Clontech, Palo Alto, CA)
and was transferred from Escherichia coli DH5 into
Agrobacterium tumefaciens LBA4404 via three-way mating with an E. coli strain containing a mobilizing plasmid, pRK2013.
Transformation of Arabidopsis (ecotype Wassilewskija) was performed as
previously described (Valvekens et al., 1988; Benfey et al., 1989).
T2 seeds of transgenic Arabidopsis were
germinated on GM containing 20 µM kanamycin at 22°C.
Whole plants were transferred to chromatography paper (3MM, Whatman)
for dehydration stress or to Petri dishes containing distilled water,
GM liquid medium, and 0.09 M L- or D-Pro and incubated under dim light (100 lux) for 24 h. To analyze GUS activity in germinating seeds, the transgenic plants
were grown on sterilized filter paper soaked with distilled water or GM
solution under continuous light (3000 lux).
Assay of GUS Activity and Histochemistry
GUS activity was assayed in whole-plant extracts by fluorimetric
determination of the production of 4-methylumbelliferone from the
glucuronide precursor using a standard protocol (Jefferson et al.,
1986). Histochemical localization of GUS activity was performed by
incubating whole transgenic plants in 1 mM
5-bromo-4-chloro-3-indolyl glucuronide at 37°C for 3 h to
overnight, fixing, and then incubating in a 50% to 100% ethanol
series (Nakashima et al., 1997).
| |
RESULTS |
Analysis of the Expression of the ProDH
Gene in Arabidopsis Plants
Induction of the ProDH gene in Arabidopsis plants by
dehydration or transfer of plants from agar plates to various solutions was analyzed by northern-blot analysis (Fig.
1). Expression of the ProDH
gene was transiently induced within 1 h after dehydration in
Arabidopsis plants, as reported by Kiyosue et al. (1996). The transient
expression of the gene was observed when the plants were transferred
from GM agar plates to hydroponic conditions in GM liquid medium (Fig.
1). These results indicated that during the dehydration treatment the
transient expression may have been caused by touching or wounding the
plants when they were transferred from the GM agar plates. The gene was
strongly induced within 1 h after the initiation of incubation,
when the plants were transferred from the same GM agar plates to
hydroponic conditions in deionized water or GM  Suc. The
osmolarity of deionized water and GM Suc was lower than that of
GM (Fig. 1). Incubation in GM Suc with 9% PEG (PEG 4000; final
osmolarity about 185 mmol/L) gave a similar result to that of GM (data
not shown). These results indicate that the high-level expression of
the ProDH gene might be due to hypoosmolarity.
View larger version (53K):
[in this window]
[in a new window]
| Figure 1.
Northern-blot analysis of the expression of the
ProDH gene in 4-week-old Arabidopsis plants after
several treatments. Each lane was loaded with 10 µg of total RNA from
3- to 4-week-old Arabidopsis plants grown in GM agar plates that were
dehydrated (Dry), transferred to hydroponic conditions for 24 h in
deionized water (DW) or in GM with and without Suc, with 0.9 M or 0.26 M L-Pro, and with 0.9 M
or 0.26 M D-Pro. Numbers above each lane indicate the
number of hours after the initiation of treatment prior to the
isolation of RNA. The osmolarity of each solution is shown at the right
side of the blots. Data represent means ± SE
(n = 3).
|
|
We previously reported that the level of ProDH mRNA in Arabidopsis
plants was strongly affected by incubation with GM containing 0.26 M L- or D-Pro instead of 0.09 M Suc (Kiyosue at al., 1996). Since the osmolarity of the
medium containing 0.09 M Pro (approximately 185 mmol
kg 1; Fig. 1) was the same as that of the
control GM solution, we examined, by northern-blot analysis, the
induction of the ProDH gene by incubation with GM containing
0.09 M Pro. When the Arabidopsis plants were transferred
from agar plates to hydroponic growth in the 0.09 M
L-Pro or D-Pro solution, the ProDH
gene was significantly induced within 1 h, although the induction
level of the ProDH mRNA by 0.09 M Pro was lower
than that by 0.26 M Pro (Fig. 1). Thus, we confirmed the
effect of L- and D-Pro in the induction of the
ProDH gene in Arabidopsis plants.
Cloning and Sequence Analysis of the Promoter Region of the
Arabidopsis ProDH Gene
We screened a genomic library prepared from Arabidopsis plants
using the insert DNA fragment of the ERD5 cDNA and isolated a 1.4-kb
DNA fragment. The nucleotide sequence of this genomic DNA was
determined (Fig. 2). The 5 end of the
transcript was determined by primer extension (data not shown). Two
bands appeared by primer-extension assay at the +1 (adenine) and +2
(cytosine) sites. Since the band in the +1 site was thicker than that
in the +2 site, we determined that the transcription start site was at
the +1 site. The initiation codon is 123 nucleotides downstream from
the site of initiation of transcription. A typical TATA-box sequence is located at position 37 (TATAAA). A number of sequence motifs have been identified that may have a role in the regulation of
transcription of plant genes. The upstream region of the
ProDH gene was searched for cis-acting elements.
We found two G-box-like motifs (ACGTG at 46 and 72) similar to the
G-box sequence (CACGTG; Schindler et al., 1992; Shinozaki and
Yamaguchi-Shinozaki, 1997) and the ABA-responsive element sequence
(PyACGTGGC; Marcotte et al., 1989; Yamaguchi-Shinozaki et al., 1989;
Shinozaki and Yamaguchi-Shinozaki, 1997). Moreover, we found four
putative motifs (TAACAG at 83, CCGTTG at 186, TAGTTA at 1132, and
TAGTTG at 1179) that resembled a MYB recognition site (PyAACNPu;
Biedenkapp et al., 1988; Nakagoshi et al., 1990; Shinozaki and
Yamaguchi-Shinozaki, 1997) and two putative motifs (CACATG at 105 and
745) that resembled a MYC recognition site (CANNTG; Murre et al.,
1989; Shinozaki and Yamaguchi-Shinozaki, 1997).
View larger version (47K):
[in this window]
[in a new window]
| Figure 2.
The nucleotide sequence of the promoter region of
the ProDH gene. The 5 end of the transcript is
indicated in the nucleotide sequence as position +1. A putative TATA
box (TATAA), G-box-like sequence (ACGTG), MYB-like sequence (PyAACNPu),
and MYC-like sequence (CANNTG) are underlined. The nucleotide sequence
was analyzed with the GENETYX software system (Software Development,
Tokyo, Japan).
|
|
Expression Analysis of GUS Reporter Gene Driven by the
ProDH Promoter in Transgenic
Arabidopsis Plants
To examine whether the cis-acting elements
involved in the hypoosmolarity and Pro-responsive expression of the
ProDH gene are located in the isolated 1.4-kb DNA fragment,
a chimeric gene consisting of the ProDH promoter region
(1393 to +122, Fig. 2) fused to the GUS reporter gene was constructed
and introduced into Arabidopsis plants through A. tumefaciens. Stable transformant lines were obtained. Figure
3 presents the analysis of the expression of the GUS reporter gene in three independent, regenerated,
T2 transgenic Arabidopsis plants
(n = 5). GUS activity was repressed within 24 h of
dehydration of Arabidopsis plants. Incubation with deionized water and
treatment with 0.09 M L-Pro or
D-Pro increased GUS activity, whereas GM incubation did
not. Incubation with 100 µM ABA or
GA3 had no effect on the expression of GUS
activity (data not shown). These data suggest that the isolated
5-upstream region of the ProDH gene contains
cis-element(s) involved in the dehydration-repressive,
hypoosmolarity-, and Pro-inducible expression. Dose-response analysis
of GUS activity revealed that incubation in 0.001 to 0.09 M
L-Pro or D-Pro induced expression of the
ProDH promoter in the three independent, regenerated
transgenic Arabidopsis plants (n = 5; Fig.
4).
View larger version (18K):
[in this window]
[in a new window]
| Figure 3.
Stress-inducible GUS activity in 4-week-old
T2 transgenic Arabidopsis plants, each of which carries
1.4-kbp of the ProDH promoter-GUS fusion gene. Some
plants were used immediately for assay of GUS activity (Control), and
the other plants were treated for 24 h by dehydration (Dry) or by
hydroponic growth in distilled water (DW), GM, or GM with 0.09 M
L-Pro or D-Pro. The values of GUS activity are the
averages of values obtained from five plants of three independent
transformant lines. Bars indicate SE.
|
|
View larger version (31K):
[in this window]
[in a new window]
| Figure 4.
Dose-response analysis of Pro-inducible GUS
activity in T2 transgenic Arabidopsis plants, each of which
carries the ProDH promoter-GUS fusion gene.
One-month-old aseptic transgenic Arabidopsis plants were transferred to
GM liquid medium with 0 (GM), 0.001, 0.005, 0.01, 0.05, or 0.09 M L-Pro or D-Pro, instead of the
same molar amount of Suc, and incubated for 24 h. Some plants were
used immediately for assay of GUS activity (Control). The values of GUS
activity are the averages of values obtained from five plants of three
independent transformant lines. Bars indicate SE.
|
|
We examined the localization of GUS reporter gene expression under
control of the ProDH promoter in transgenic Arabidopsis plants. Weak GUS activity was observed by histochemical analysis in
unbolting, whole transgenic Arabidopsis seedlings 2 weeks after germination without any treatment (Fig.
5A). GUS activity in the leaves was
relatively low (Fig. 5B). GUS expression was detected in the main roots
(Fig. 5C) and some, but not all, root tips (Fig. 5D). After 0.09 M L-Pro incubation, intense GUS staining was
observed in the veins and hydathodes of leaves (Fig. 5E) and in the
lateral and main roots (Fig. 5G), especially in the root vascular
cylinder and meristem (Fig. 5H). Examination of tissue sections of
leaves revealed that the strong expression in veins was not due to a smaller cell volume than in mesophyll cells but, rather, to high expression in vascular cells (data not shown). The same expression pattern was found in transformants after 0.09 M
D-Pro or water incubation (data not shown). L-
and D-Pro had a stronger effect on ProDH gene
expression than water in the same plant tissues.
View larger version (96K):
[in this window]
[in a new window]
| Figure 5.
Histochemical localization of GUS activity in 10- to 14-d-old transgenic Arabidopsis plants containing the
ProDH promoter-GUS fusion gene. A, Overview of GUS
activity, showing weak activity in rosette plants stained for
6 h. B, Little GUS activity in leaves stained for 2 h. C,
Strong GUS staining in main root stained for 6 h. D, GUS staining
in some root primordia stained for 2 h. E through H, GUS staining
in plants after 24 h of L-Pro treatment. E, Overview
of GUS activity in roots and leaves stained for 6 h. F, Strong GUS
staining in veins and hydathodes (arrowheads) of leaves stained for
2 h. G, GUS staining in root tip and veins stained for 6 h.
H, Strong GUS staining in root tips stained for 2 h. Bars = 200 µm.
|
|
In 8-week-old transgenic Arabidopsis plants, expression of GUS was
observed in the flowers (Fig. 6A) without
any treatment, especially in the pollen and the stigmas with pollen
(Fig. 6B), the stigma of immature siliques (shorter than 5 mm), the
ovules, and the abscission zone of the petals and sepals (base of the silique; Fig. 6C), which is a vestige of the stigma. Observation by
microscopy of the flower staining showed that GUS activity around the
stigmas in the flowers and the tips of the siliques was due to pollen
attached to the stigmas rather than to the stigmas themselves (data not
shown). The stigma surface of the longer siliques (longer than 5 mm),
but not their ovules, showed GUS staining. Mature siliques (longer than
7 mm) showed no GUS staining. However, high GUS activity was observed
in seeds of mature siliques that had been cut to facilitate the
penetration of 1 mM 5-bromo-4-chloro3-indolyl glucuronide (Fig. 6D). These results indicate that the seed coats inhibited penetration of the GUS substrate. In the seeds high GUS
activity staining was observed in the tips of the cotyledons and in the
shoot and root meristem.
View larger version (78K):
[in this window]
[in a new window]
| Figure 6.
Histochemical localization of GUS activity in
3-month-old flowering transgenic Arabidopsis plants and the mature seed
containing the ProDH promoter-GUS fusion gene. A, Flower
stained overnight. B, Strong GUS staining in pollen grains and stigmas
of flowers stained overnight. C, Silique showing strong GUS staining in
the stigma, ovules, and abscission zone stained overnight. D,
Longitudinal sections of the mature seed showing strong GUS staining,
stained for 4 h. az, Abscission zone; co, cotyledon; ov, ovule;
pg, pollen grain; rm, root meristem; sg, stigma; and sm, shoot
meristem. Bars = 200 µm.
|
|
When transgenic T2 seeds were germinated on
filter paper soaked with water, GUS activity increased in the
germinating seeds under hypoosmolar conditions (Fig.
7). Strong GUS activity was observed
mainly in the root tips and veins of germinating seeds (Fig. 7 B).
However, when transgenic plants were grown on filter paper soaked with
GM solution, GUS activity did not increase during germination (Fig. 7),
and only weak GUS activity was observed in the root tips of germinating
seeds. The germination process of the seeds in the GM solution was
slower than that in water.
View larger version (38K):
[in this window]
[in a new window]
| Figure 7.
Changes of GUS activity during germination of
transgenic Arabidopsis plants containing the ProDH
promoter-GUS fusion gene. A, GUS activity in germinating T2
transgenic Arabidopsis plants, each of which carries the gene. The
transgenic plants were grown on sterilized filter papers soaked with
distilled water (DW) or GM solution. The values of GUS activity are
averages of two repeats of three independent transformant lines
(n = 5-10). Bars indicate SE. B,
Histochemical localization of GUS activity in germinating transgenic
Arabidopsis plants containing the ProDH
promoter-GUS fusion gene. M, Mature seed showing strong GUS staining,
stained for 6 h; DW 1d to DW 4d, germinating seeds on a filter
paper soaked with distilled water at 1, 2, 3, and 4 d after
planting (stained for 6 h). Bars = 200 µm.
|
|
| |
DISCUSSION |
We have shown, by northern-blot analysis, that the
ProDH gene is repressed by dehydration but induced by
rehydration and by L- and D-Pro treatment
(Kiyosue et al., 1996). We analyzed the expression of the
ProDH gene and found that it is up-regulated by
hypoosmolarity and by Pro treatment (Fig. 1). The induction of
ProDH gene expression under rehydration after 10 h of
dehydration is caused not only by accumulated Pro but also by
hypoosmolarity. Therefore, the ProDH gene is controlled by
three different factors: up-regulation by hypoosmolarity and Pro and
down-regulation by dehydration stress.
We then isolated a 1.4-kb promoter region of the ProDH gene
and analyzed the function of the promoter using transgenic plants with
the 1.4-kb ProDH promoter-GUS fusion gene. We demonstrated that GUS activity driven by the 1.4-kb promoter region was depressed by
dehydration (Fig. 3). Northern-blot hybridization and promoter analysis
using transgenic plants revealed that the 1.4 kb of the ProDH promoter was activated by hypoosmolarity treatment
with GM Suc solution and water (Figs. 1 and 3). Because the
osmolarity of these solutions was lower than the control medium, GM,
hypoosmolarity induces ProDH gene expression. Kiyosue et al.
(1996) previously reported that a slight induction in ProDH
gene expression was detected when the plants were incubated in GM Suc or water for 10 h. However, analysis of the
ProDH mRNA by northern-blot analysis revealed that
accumulation of the ProDH mRNA began within 1 h after
the transfer of the plants from the agar plates to GM Suc or
water solutions, reached a maximum after 2 h of incubation, and
then decreased. Northern analysis and analysis using transgenic Arabidopsis also revealed that the promoter of the ProDH
gene was up-regulated by 0.001 to 0.09 M Pro (Fig. 4). The
dose dependency of the promoter was consistent with the
northern-analysis results of Kiyosue et al. (1996). GUS activity was
not detected in dehydrated Arabidopsis plants (Fig. 3). These results
indicate that the 1.4-kb promoter region contains all of the
cis-acting elements involved in up-regulation by Pro and
hypoosmolarity and down-regulation by dehydration.
Kiyosue et al. (1996) reported that 0.26 M
D-Pro induced more stable expression of the
ProDH gene than 0.26 M L-Pro in
Arabidopsis. Figure 1 also suggested that 0.09 M
D-Pro treatment for 24 h results in the stronger
induction of the ProDH gene expression than 0.09 M L-Pro. However, GUS activity data in Figures
3 and 4 contradict this observation, showing that L-Pro
induced a higher level of the ProDH promoter activity. This
contradiction may have been due to an experimental variation in the GUS
activity caused by differences of insertion positions of the
ProDH promoter-GUS fusion gene in transgenic lines.
Alternatively, it could have been due to posttranscriptional effects in
synthesizing or degradation of the GUS proteins by exogenous amino
acids, especially D-Pro.
When the Arabidopsis plants were treated with water or Pro, the
ProDH gene was expressed at high levels in all vegetative tissues analyzed (Fig. 5). Highest expression was found in the veins
and hydathodes of leaves and root tips under water or Pro supply (Fig.
5, E-H). Histochemical analysis showed that Pro has a stronger effect
on the ProDH gene expression than does water in the same
plant tissues. Although Pro is not supplied from roots in natural
conditions, it is reasonable to assume that excess Pro in cells would
be degraded by ProDH. It is suspected that the applied Pro may be
transported to almost all parts of plants by Pro transporters such as
ProT1 and ProT2 (Rentsch et al., 1996), and that it triggers
ProDH gene expression. When alfalfa plants are exposed to
water-deficit conditions, Pro accumulates in the phloem sap (Girousse
et al., 1996). Expression of the ProDH gene in veins of
rehydrated plants indicated that vascular cells convert the accumulated
Pro under water-deficit conditions for Glu by ProDH in rehydrated
plants. Hydathodes are secretory structures that remove water from the
interior of a leaf and deposit it on the leaf surface. Water is forced
out of the hydathodes by hydrostatic pressure, and an osmolarity
gradient may be established inside the hydathodes. The level of Pro,
which is controlled by synthesis and degradation processes, may play a
role in the osmolarity gradient of hydathodes. Alternatively,
hydathodes may have a role in reabsorption of nutrients, including Pro
from vascular bundles, and degrade the Pro by ProDH. Expression in root
meristems indicates that ProDH might play a role in catabolism of Pro
to amino acids, as well as nitrogen, carbon, energy, and reducing power
for elongation of roots in rehydrated plants.
Under normal growth conditions, the ProDH gene was expressed
at low levels in all of the vegetative tissues analyzed (Fig. 5, A-D).
High levels of expression were found in reproductive tissues such as
pollen grains and seeds (Fig. 6). Pro has been found to accumulate in
tissues such as florets and seeds, which have a low water content,
whereas tissues such as rosette leaves, which have a high water
content, contain low levels of Pro (Chiang and Dandekar, 1995). In
Arabidopsis reproductive tissues (florets and seeds), Pro represents
17% to 26% of total free amino acids, whereas in vegetative tissues
(rosette leaves and roots), Pro contributes only 1% to 3%.
Accumulation of Pro was noted in the inflorescence and siliques of
Brassica napus (Flasinski and Rogozinska, 1985). In
naturally desiccated tissues such as pollen, a high concentration of
Pro is correlated with protection against pollen germination at
unfavorable temperatures (Zhang and Croes, 1983). The elevated
expression of the ProDH gene in pollen grains and seeds in
Arabidopsis is consistent with an increased accumulation of Pro in
these organs. Pro in pollens and seeds is considered to be
dehydrogenated by ProDH to supply Glu or derived compounds and energy
for their growth and development.
When transgenic plants containing the ProDH promoter-GUS
fusion were germinated in water, GUS activity increased (Figs. 7 and
8). However, when transgenic plants were germinated in GM, GUS activity
did not increase. These data indicate that the Pro in seeds might be
oxidized to control the osmolarity of seed cells under hypoosmolarity,
and used for seed germination and elongation under conditions of
insufficient nutrients.
Mutant analysis indicates that the synthesis and sensitivity of
GA3 are essential for seed germination and
elongation growth in Arabidopsis (Koornneef and Karssen, 1994). Because
the expression of the ProDH gene is not affected by GA
(Kiyosue et al., 1996; and our results using the transgenic plants),
ProDH gene expression during seed germination in water may
be due to hypoosmolarity. Thus, ProDH might play a role in supplying
nutrients and energy from accumulated Pro for development and
germination of reproductive organs such as seeds.
In higher plants Pro is synthesized via two routes, from Orn and from
Glu. In young Arabidopsis plantlets, the Orn pathway, together with the
Glu pathway, plays an important role in Pro accumulation during osmotic
stress (Roosens et al., 1998). In adult Arabidopsis plantlets, the free
Pro increase is mainly due to the activity of the enzymes of the Glu
pathway (Yoshiba et al., 1997; Roosens et al., 1998). Genes encoding
P5CS and P5CR for the Glu pathway have been cloned in Arabidopsis
(Savouré et al., 1995; Strizhov et al., 1997; Yoshiba et al.,
1997). P5CS has been shown to catalyze the limiting step in Pro
accumulation in response to osmotic stress in adult Arabidopsis plants
(Strizhov et al., 1997; Yoshiba et al., 1997).
Recently, Hua et al. (1997) reported that the promoter of
At-P5R, a gene encoding P5CR in Arabidopsis, directs strong
GUS activity in root tips, the shoot meristem, guard cells, hydathodes, veins, pollen grains, ovules, and developing seeds. Among these sites,
the root tips, hydathodes, veins, pollen, and seeds overlap the GUS
expression sites directed by the ProDH gene, although the
timing of the expression of the ProDH gene is different from that of the P5CR gene. GUS staining in the root tips,
hydathodes, and veins of transformants with the P5CR
promoter-GUS construct was observed under normal conditions (Hua et
al., 1997), whereas GUS staining of transformants with the
ProDH promoter-GUS construct was strong under high-Pro or
hypoosmolar conditions (Fig. 5). Osmolarity of plant tissues may be
controlled by the synthesis and degradation of Pro in these sites.
During seed ripening GUS activity driven by the P5CR
promoter generally decreased, and when the siliques reached 1 cm in
length, no staining could be observed (Hua et al., 1997). This pattern
is similar to that in the siliques of transformants containing the
ProDH promoter-GUS fusion gene (Fig. 6C). Although seeds
containing the P5CR promoter-GUS fusion gene were stained
after they had been cut apart and treated with 1 mM
5-bromo-4-chloro-3-indolyl glucuronide, no staining was observed (Hua
et al., 1997), whereas mature seeds with the ProDH
promoter-GUS fusion gene were stained (Fig. 6D). Germinating seeds with
the ProDH promoter-GUS fusion gene were also stained under
hypoosmolarity (Fig. 7).
These data indicate that the P5CR gene for Pro synthesis is
expressed during the early developmental stage of seeds and that the
ProDH gene for Pro degradation is expressed during the later developmental stage and during germination of seeds. The
ProDH gene was expressed in veins and root tips when plants
were exposed to water or Pro, whereas the P5CR gene was
expressed in these sites in the absence of stress. However, higher
levels of mRNA of the Pro-transporter ProT1 were detected in
flowers, and the expression was down-regulated during development in
Arabidopsis (Rentsch et al., 1996). We presume that the Pro accumulated
in flowers might be synthesized and/or transported at an early
developmental stage and degraded to Glu by ProDH for use as a source of
nutrients and energy for ripening and germination of pollen and seeds,
respectively. We previously reported that P5CS is the key enzyme for
Pro biosynthesis in Arabidopsis (Yoshiba et al., 1995). Histochemical
analysis of the promoter of the P5CS gene will give us more
information about the role of Pro in the development of plants and
their acclimation to environmental stress.
This is the first report, to our knowledge, of a promoter that is
negatively regulated by dehydration and up-regulated by hypoosmolarity
and Pro. The isolated promoter region is also developmentally regulated, especially in reproductive organs. Detailed analysis of the
promoter is required to clarify the molecular mechanism of
dehydration-rehydration and Pro metabolism in plants.
| |
FOOTNOTES |
1
This research was supported in part by the
Program for Promotion of Basic Research Activities for Innovative
Biosciences, the Human Frontier Science Program, the Special
Coordination Fund of the Science and Technology Agency, and a grant-in
aid from the Ministry of Education, Science and Culture of Japan.
2
Present address: National Institute for Basic
Biology, Myodaiji-cho, Okazaki 444, Japan.
*
Corresponding author; e-mail kazukoys{at}jircas.affrc.go.jp; fax
81-298-38-6643.
Received May 26, 1998;
accepted August 18, 1998.
The accession number for the DNA sequence reported in this article is
AB008810.
| |
ABBREVIATIONS |
Abbreviations:
P5CR,  1-pyrroline-5-carboxylate
reductase.
P5CS, 1-pyrroline-5-carboxylate synthetase.
| |
ACKNOWLEDGMENTS |
We thank Ms. A. Konishi and Ms. S. Yoshida (Japan International
Research Center for Agricultural Sciences) for their excellent technical assistance. We also thank Dr. Y. Yoshiba (Advanced Research Laboratory, Hitachi Ltd., Japan) for helpful discussions.
| |
LITERATURE CITED |
Ahmad I,
Hellebust JA
(1988)
The relationship between inorganic nitrogen metabolism and proline accumulation in osmoregulatory responses of two euryhaline microalgae.
Plant Physiol
88:
348-354
[Abstract/Free Full Text]
Benfey PN,
Ren L,
Chua N-H
(1989)
The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns.
EMBO J
8:
2195-2202
[Web of Science][Medline]
Biedenkapp H,
Borgmeyer U,
Sippel AE,
Klempnauer K-H
(1988)
Viral myb oncogene encodes a sequence-specific DNA-binding activity.
Nature
335:
835-837
[CrossRef][Medline]
Boyer JS
(1982)
Plant productivity and environment.
Science
218:
443-448
[Abstract/Free Full Text]
Chiang H-H,
Dandekar AM
(1995)
Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation.
Plant Cell Environ
18:
1280-1290
Delauney AJ,
Verma DPS
(1993)
Proline biosynthesis and osmoregulation in plants.
Plant J
4:
215-223
Flasinski S,
Rogozinska J
(1985)
Effect of water deficit on proline accumulation, protein and chlorophyll content during flowering and seed formation in winter rape.
Acta Agrobot
38:
11-21
Girousse C,
Bournoville R,
Bonnemain J-L
(1996)
Water deficit-induced changes in concentrations in proline and some other amino acids in the phloem sap of alfalfa.
Plant Physiol
111:
109-113
[Abstract]
Hua X-J,
Van de Cotte B,
Montagu MV,
Verbruggen N
(1997)
Developmental regulation of pyrroline-5-carboxylate reductase gene expression in Arabidopsis.
Plant Physiol
114:
1215-1224
[Abstract]
Igarashi Y,
Yoshiba Y,
Sanada Y,
Yamaguchi-Shinozaki K,
Wada K,
Shinozaki K
(1997)
Characterization of the gene for 1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.
Plant Mol Biol
33:
857-865
[CrossRef][Web of Science][Medline]
Jefferson RA,
Burgess SM,
Hirsh D
(1986)
-Glucuronidase from Escherichia coli as a gene-fusion marker.
Pro Natl Acad Sci USA
83:
8447-8451
[Abstract/Free Full Text]
Kiyosue T,
Yamaguchi-Shinozaki K,
Shinozaki K
(1993a)
Characterization of cDNA for a dehydration-inducible gene that encodes a Clp A, B-like protein in Arabidopsis thaliana L.
Biochem Biophys Res Commun
196:
1214-1220
[CrossRef][Web of Science][Medline]
Kiyosue T,
Yamaguchi-Shinozaki K,
Shinozaki K
(1993b)
Characterization of two cDNAs (ERD11 and ERD13) for dehydration-inducible genes that encode putative glutathione S-transferases in Arabidopsis thaliana L.
FEBS Lett
335:
189-192
[CrossRef][Web of Science][Medline]
Kiyosue T,
Yamaguchi-Shinozaki K,
Shinozaki K
(1994)
Cloning of cDNA for genes that are early responsive to dehydration-stress (ERDs) in Arabidopsis thaliana L.: identification of three ERDs as HSP cognate genes.
Plant Mol Biol
25:
791-798
[CrossRef][Web of Science][Medline]
Kiyosue T,
Yoshiba Y,
Yamaguchi-Shinozaki K,
Shinozaki K
(1996)
A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis.
Plant Cell
8:
1323-1335
[Abstract]
Koornneef M,
Karssen CM
(1994)
Seed dormancy and germination.
In
EM Meyerowitz,
CR Somerville,
eds, Arabidopsis.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 313-334
Marcotte WR Jr,
Russell SH,
Quatrano RS
(1989)
Abscisic acid response sequences from the Em gene of wheat.
Plant Cell
1:
969-976
[Abstract/Free Full Text]
Murre C,
McCaw PS,
Baltimore D
(1989)
A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, myoD, and myc proteins.
Cell
56:
777-783
[CrossRef][Web of Science][Medline]
Nakagoshi H,
Nagase T,
Kanei-Ishii C,
Ueno Y,
Ishii S
(1990)
Binding of the c-myb proto-oncogene product to the simian virus 40 enhancer stimulates transcription.
J Biol Chem
265:
3479-3483
[Abstract/Free Full Text]
Nakashima K,
Kiyosue T,
Yamaguchi-Shinozaki K,
Shinozaki K
(1997)
Plant J
12:
851-861
[CrossRef][Web of Science][Medline]
Peng Z,
Lu Q,
Verma DPS
(1996)
Reciprocal regulation of 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants.
Mol Gen Genet
253:
334-341
[Web of Science][Medline]
Rentsch D,
Hirner B,
Schmelzer E,
Frommer WB
(1996)
Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.
Plant Cell
8:
1437-1446
[Abstract]
Roosens NHCJ,
Thu TT,
Iskander HM,
Jacobs M
(1998)
Isolation of the ornithine- -aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana.
Plant Physiol
117:
263-271
[Abstract/Free Full Text]
Savouré A,
Jaoua S,
Hua X-J,
Ardiles W,
Montagu MV,
Verbruggen N
(1995)
Isolation, characterization, and chromosomal location of a gene encoding the 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana.
FEBS Lett
372:
13-19
[CrossRef][Web of Science][Medline]
Schindler U,
Menkens AE,
Beckmann H,
Ecker JR,
Cashmore AR
(1992)
Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins.
EMBO J
11:
1261-1273
[Web of Science][Medline]
Shinozaki K,
Yamaguchi-Shinozaki K
(1996)
Molecular responses to drought and cold stress.
Curr Opin Biotechnol
7:
161-167
[CrossRef][Web of Science][Medline]
Shinozaki K,
Yamaguchi-Shinozaki K
(1997)
Gene expression and signal transduction in water-stress response.
Plant Physiol
115:
327-334
[CrossRef][Web of Science][Medline]
Smirnoff N,
Cumbes QJ
(1989)
Hydroxyl radical scavenging activity of compatible solutes.
Phytochemistry
28:
1057-1060
[CrossRef][Web of Science]
Strizhov N,
Ábrahám E,
Ökrész L,
Blickling S,
Zilberstein A,
Schell J,
Koncz C,
Szabados L
(1997)
Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis.
Plant J
12:
557-569
[CrossRef][Web of Science][Medline]
Valvekens D,
Montagu MV,
Lijsebettens MV
(1988)
Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.
Proc Natl Acad Sci USA
85:
5536-5540
[Abstract/Free Full Text]
Verbruggen N,
Hua X-J,
May M,
Montagu MV
(1996)
Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.
Proc Natl Acad Sci USA
93:
8787-8791
[Abstract/Free Full Text]
Walton EF,
Clark CJ,
Boldingh HL
(1991)
Effects of hydrogen cyanamide on amino acid profiles in kiwifruit buds during budbreak.
Plant Physiol
97:
1256-1259
[Abstract/Free Full Text]
Yamaguchi-Shinozaki K,
Mundy J,
Chua N-H
(1989)
Four tightly linked rab genes are differentially expressed in rice.
Plant Mol Biol
14:
29-39
Yoshiba Y,
Kiyosue T,
Katagiri T,
Ueda H,
Mizoguchi T,
Yamaguchi-Shinozaki K,
Wada K,
Harada Y,
Shinozaki K
(1995)
Correlation between the induction of a gene for 1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress.
Plant J
7:
751-760
[CrossRef][Web of Science][Medline]
Yoshiba Y,
Kiyosue T,
Nakashima K,
Yamaguchi-Shinozaki K,
Shinozaki K
(1997)
Regulation of the level of proline as an osmolyte in plants under water stress.
Plant Cell Physiol
38:
1095-1102
[Abstract/Free Full Text]
Zhang H-Q,
Croes AF
(1983)
Protection of pollen germination from adverse temperature: a possible role for proline.
Plant Cell Environ
6:
471-476
This article has been cited by other articles:
|
|
|
|
 
F. Anzala, M.-C. Morere-Le Paven, S. Fournier, D. Rondeau, and A. M. Limami
Physiological and molecular aspects of aspartate-derived amino acid metabolism during germination and post-germination growth in two maize genotypes differing in germination efficiency
J. Exp. Bot.,
February 1, 2006;
57(3):
645 - 653.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yamada, H. Morishita, K. Urano, N. Shiozaki, K. Yamaguchi-Shinozaki, K. Shinozaki, and Y. Yoshiba
Effects of free proline accumulation in petunias under drought stress
J. Exp. Bot.,
July 1, 2005;
56(417):
1975 - 1981.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Deuschle, D. Funck, G. Forlani, H. Stransky, A. Biehl, D. Leister, E. van der Graaff, R. Kunze, and W. B. Frommer
The Role of {Delta}1-Pyrroline-5-Carboxylate Dehydrogenase in Proline Degradation
PLANT CELL,
December 1, 2004;
16(12):
3413 - 3425.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Sakamoto, K. Maruyama, Y. Sakuma, T. Meshi, M. Iwabuchi, K. Shinozaki, and K. Yamaguchi-Shinozaki
Arabidopsis Cys2/His2-Type Zinc-Finger Proteins Function as Transcription Repressors under Drought, Cold, and High-Salinity Stress Conditions
Plant Physiology,
September 1, 2004;
136(1):
2734 - 2746.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Contento, S.-J. Kim, and D. C. Bassham
Transcriptome Profiling of the Response of Arabidopsis Suspension Culture Cells to Suc Starvation
Plant Physiology,
August 1, 2004;
135(4):
2330 - 2347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Satoh, Y. Fujita, K. Nakashima, K. Shinozaki, and K. Yamaguchi-Shinozaki
A Novel Subgroup of bZIP Proteins Functions as Transcriptional Activators in Hypoosmolarity-Responsive Expression of the ProDH Gene in Arabidopsis
Plant Cell Physiol.,
March 15, 2004;
45(3):
309 - 317.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Seki, M. Satou, T. Sakurai, K. Akiyama, K. Iida, J. Ishida, M. Nakajima, A. Enju, M. Narusaka, M. Fujita, et al.
RIKEN Arabidopsis full-length (RAFL) cDNA and its applications for expression profiling under abiotic stress conditions
J. Exp. Bot.,
January 2, 2004;
55(395):
213 - 223.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Nanjo, M. Fujita, M. Seki, T. Kato, S. Tabata, and K. Shinozaki
Toxicity of Free Proline Revealed in an Arabidopsis T-DNA-Tagged Mutant Deficient in Proline Dehydrogenase
Plant Cell Physiol.,
May 15, 2003;
44(5):
541 - 548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Limami, C. Rouillon, G. Glevarec, A. Gallais, and B. Hirel
Genetic and Physiological Analysis of Germination Efficiency in Maize in Relation to Nitrogen Metabolism Reveals the Importance of Cytosolic Glutamine Synthetase
Plant Physiology,
December 1, 2002;
130(4):
1860 - 1870.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Satoh, K. Nakashima, M. Seki, K. Shinozaki, and K. Yamaguchi-Shinozaki
ACTCAT, a Novel cis-Acting Element for Proline- and Hypoosmolarity-Responsive Expression of the ProDH Gene Encoding Proline Dehydrogenase in Arabidopsis
Plant Physiology,
October 1, 2002;
130(2):
709 - 719.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. RAYMOND and N. SMIRNOFF
Proline Metabolism and Transport in Maize Seedlings at Low Water Potential
Ann. Bot.,
June 15, 2002;
89(7):
813 - 823.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Mani, B. Van de Cotte, M. Van Montagu, and N. Verbruggen
Altered Levels of Proline Dehydrogenase Cause Hypersensitivity to Proline and Its Analogs in Arabidopsis
Plant Physiology,
January 1, 2002;
128(1):
73 - 83.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Medina, R. Catalá, and J. Salinas
Developmental and Stress Regulation of RCI2A and RCI2B, Two Cold-Inducible Genes of Arabidopsis Encoding Highly Conserved Hydrophobic Proteins
Plant Physiology,
April 1, 2001;
125(4):
1655 - 1666.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
F. Hayashi, T. Ichino, M. Osanai, and K. Wada
Oscillation and Regulation of Proline Content by P5CS and ProDH Gene Expressions in the Light/Dark Cycles in Arabidopsis thaliana L.
Plant Cell Physiol.,
October 1, 2000;
41(10):
1096 - 1101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hellmann, D. Funck, D. Rentsch, and W. B. Frommer
Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application
Plant Physiology,
June 1, 2000;
123(2):
779 - 789.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
H. Hellmann, D. Funck, D. Rentsch, and W. B. Frommer
Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application
Plant Physiology,
February 1, 2000;
122(2):
357 - 368.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction